Natural killer T (NKT) cells are a subset of T cells that co-express an αβ T cell receptor (TCR), but also express a variety of molecular markers that are typically associated with NK cells. They differ from conventional αβ T cells in that the diversity of their TCRs is much more limited and in that they recognize lipids and glycolipids presented by the major histocompatibility complex (MHC)-like molecules, such as CD1d molecules, part of the CD1 family of antigen presenting molecules, rather than peptide-MHC complexes (Brigl, M et al., Annu. Rev. Immunol., 22:817-890, 2004). NKT cells are stimulated by APC via CD1d-TCR interactions and rapidly produce many predominantly Th1 or Th2 type cytokines, such as interferon-γ (IFN-γ, Th1) and interleukin-4 (IL-4, Th2), which play an important role in the activation of the cells responsible for innate and adaptive immune responses. The balance of Th1/Th2 cytokine responses is known to play an important role in orchestrating immune response properties (Chen et al., J. Immunol., 159: 2240, 1997; Wilson et al., Proc. Natl. Acad. Sci. U.S.A., 100: 10913, 2003).
α-Galactosylceramide (α-GalCer), a structurally modified derivative of the extract agelasphin from marine sponges, was first discovered as a ligand for the Vα14+ T cell receptor of NKT (natural killer T cell) and shows highly potent activity (Morita, M. et al., J. Med. Chem. 38:2176-2187, 1995). Since the discovery thereof, α-GalCer has been most widely studied in the context of the biological and pharmaceutical properties of NKT cells. Many derivatives thereof have been synthesized and evaluated for structure-activity relationship (SAR) (Savage, P., et al., Chem. Soc. Rev., 35:771-779, 2006). SAR studies on the sugar moiety of α-GalCer revealed that the galactosyl group plays an important role in the activation of NKT cells through an α-GalCer/CD1d/TCR interaction. The α-anomeric linkage structure was found to completely allow α-GalCer to act as a potent and effective ligand for NKT cells (Kawano, T. et al., Science, 278:1626-1629, 1997). Also, it was reported that, whereas the hydroxide group at position 2 of the sugar moiety is most responsible for the recognition of NKT cells through TCR, chemical modification can be applied to the hydroxide groups at positions 3 and 6 (Barbieri, L. et al., Eur. J. Org. Chem., 468-473, 2004).
Modifications in fatty acid chains as well as in the sphingosine moiety are likely to bring about a change in the stability of the glycolipid-CD1d complex, thus having an influence on cytokine secretion. For example, PBS-25, an α-GalCer variant with a shorter fatty acid chain, induces the secretion of a greater amount of Th2-cytokines, compared to α-GalCer (Goff, R. D. et al., J. Am. Chem. Soc., 126:13602-13603, 2004). The removal of 9 carbons from the fatty acid chain of phytosphingosine increases the relative amounts of Th2-cytokine release by NKT cells (Miyamoto, K. et al., Nature, 413:531-534, 2001) and the presence of an aromatic ring in the fatty acid chain results in an increase in Th1 cytokine secretion (Fujio, M. et al., J. Am. Chem. Soc., 128:9022-9023, 2006). Interestingly, the substitution of the oxygen atom at the anomer position of α-GalCer with CH2 changes the pattern of cytokine secretion. An animal test showed that these C-glycoside analogs to α-GalCer can strongly induce the secretion of Th1-cytokines in NKT cells (Frank, R. W. et al., Acc. Chem. Res., 39:692-701, 2006).
The SAR of α-GalCer has recently been identified through the X-ray crystallographic structure of an α-GalCer-CD1d complex (Zajonc, D. M. et al., Nat. Immunol., 6:810-818, 2005), which shows the accurate fit of the α-GalCer fatty acid chain into the two hydrophobic grooves of CD1d. Hydrogen bonds between the surface residues of CD1d and the hydroxide groups of galactose and sphingoshine are believed to play a critical role in maintaining the accurate position and direction of α-GalCer necessary for TCR recognition. The crystalline structure of the complex of human CD1d and α-GalCer demonstrated that the amide group of α-GalCer does not form a hydrogen bond with the surface residues of CD1d (Koch, M. et al., Nat. Immunol., 6:819-826, 2005). In the crystalline structure of the complex of murine CD1d and PBS-25, the NH of the PBS-25 amide is seen to form a hydrogen bond with the α2 spiral structure of CD1d, but the carbonyl group of the amide does not, indicating that the amide group of α-GalCer may be a recognition target in contact with the TCR of NKT cells, in addition to playing a structural role in determining the three-dimensional position of the fatty acid chain.
The α-GalCer variants reported thus far can be divided into three groups: modifications in sphingosine moiety; fatty acid chain; and sugar moiety. Nowhere have partial modifications in the amide moiety of α-GalCer been reported before the present invention. Because the amide group acts to form a hydrogen bond at a binding position, the bioisosteric replacement of the α-GalCer amide moiety can provide interesting information about the biological features of NKT cells. Thanks to the different electronic and steric properties of isosteres, modification of the amide moiety results in a change in the stability of glycolipid-CD1d complex and the position of the sugar head in the binding grooves, leading to the possibility of developing galactosylceramide having more potent antigenicity. Furthermore, the bioisosteric replacement is expected to have an influence on the metabolism of α-GalCer, thus leading to a change in immune response.
Among a variety of bioisosteres of the amide moiety, 1,2,3-triazole compounds have gained increasing attention in drug discovery since the introduction of “Click” chemistry by Sharpless (Kolb, H. C. et al., Drug Discov. Today, 8:1128-1237, 2003, Kolb, H. C., et al., Angew. Chem. Int. Ed., 40:2004-2021, 2001). 1,2,3-Triazole compounds can mimic the topological and electronic features of an amide bond, providing a firm binding unit. These compounds can actively participate in hydrogen bonding and dipole-dipole interactions, thanks to the strong dipole moment. Compared to other amide compounds, however, triazole compounds are surprisingly stable to hydrolysis and in oxidation and reduction conditions. Nowhere has the introduction of 1,2,3-triazole into the ceramide moiety been reported, thus far.
Leading to the present invention, intensive and thorough research into pharmaceutically effective modified α-GalCer derivatives, conducted by the present inventors, resulted in the finding that the bioisosteric replacement of the amide moiety of α-GalCer with triazole of various fatty acid chain lengths, based on the crystal structure of a CD1d-α-GalCer complex, increases the IL-4 vs. IFN-γ bias of released cytokines.